Prokaryotes are classified into two domains called Bacteria and Archaea, the latter of which are referred to as extremophiles. The invention described herein does not encompass Archaea. Bacteria are prokaryotes that consist of a cell membrane bound by a cell wall. Exceptions are bacteria in the genera Mycoplasma, Chlamydia, and Ureaplasma, all of which lack cell walls.
With the exception of those lacking cell walls, bacteria can be divided into two large groups based on the chemical and physical properties of their cell walls by Gram staining (Madigan, et al. Brock: Biology of Microorganisms. 12th ed. 2009, Pearson/Benjamin Cummings. pp. 27-28, 77-86). Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50%-90% of cell wall), which stains purple when subjected to Gram-staining, while Gram-negative bacteria have a thinner layer containing less peptidoglycan (10% of cell wall), which stains pink. Gram-negative bacteria also have an additional outer membrane, which contains lipopolysaccharide (LPS). Lipopolysaccharides, which are endotoxins and are very toxic, elicit a strong immunogenic response in animals. Endotoxins trigger humoral enzymatic mechanisms involving the complement, clotting cascade, fibrinolytic, and kinin pathways and can cause morbidity associated with Gram-negative sepsis. Numerous pathogens are members of the domain Bacteria.
Bacterial Pathogens and Pathogenesis
In order for a pathogen to infect a host, the microorganism must first gain entrance into the host. Entrance may occur via inhalation, ingestion, or a break in the skin's integrity, to name but a few. After entrance, the microorganism must adhere to its host tissue. Adherence is often specific and the host can employ mechanisms to avoid adherence, such as epithelial and mucosal sloughing. Once adhered, the microorganism must colonize its host (i.e., grow on the adherence surface). Following colonization, pathogenic microorganisms initiate a process of invasion in which microbes grow within their hosts tissues. It is typically in this stage of invasion that pathogens cause symptoms of disease, as discussed herein.
Pathogenic bacteria are able to cause disease because they possess certain structural, biochemical, or genetic traits that render them virulent. The sum of the characteristics that allow a given bacterium to produce disease are the pathogen's determinants of virulence. Some pathogens may rely on a single determinant of virulence, such as a toxin, to cause damage to their host, which is described in more detail below. Other pathogens, such as Staphylococcus aureus, Streptococcus pyogenes and Pseudomonas aeruginosa, maintain a large repertoire of virulence determinants and, consequently, are able to produce a wider range of diseases that affect different tissues in their host.
A limited number of extracellular proteins and enzymes that are also virulence factors have been characterized, for example:
1. Diptheria toxin. Corynebacterium diphtheriae, the causative agent of diphtheria, initially infects animals via the respiratory mucosa. Upon infection, C. diphtheriae quickly consumes its host's local supply of iron, which is a co-repressor of the DT gene (Holmes (2000) J. Infec. Diseases 181:S156-S157). In response to a low-iron microenvironment, C. diphtheria secretes a 62 kD extracellular protein known as diphtheria toxin (Schmidt et al. (1991) Infection and Immunity. 59: 1899-1904). This toxin, released into the blood steam, causes the progressive deterioration of myelin sheaths in the central and peripheral nervous system leading to degenerating motor control and loss of sensation (Atkinson, Hamborsky, and Wolfe, eds. Diphtheria. In: Epidemiology and Prevention of Vaccine-Preventable Diseases. 10th ed. 2007, Washington D.C.: Public Health Foundation. pp. 59-70). Diptheria is treated with an anti-toxin that neutralizes toxin that is not bound to tissues, as well as antibiotics to prevent further transmission (Mayo Foundation for Medical Education and Research, 2009. Diphtheria: Treatment and Drugs.)
2. Clostridium toxins. C. difficile, C. botulinum, C. tetani, and C. perfringens all produce protein toxins that cause symptoms ranging from abdominal cramps, diarrhea and vomiting associated with food poisoning, gas gangrene at the site of trauma or recent surgical wounds, spasms and rigidity of voluntary muscles, and colitis. These symptoms are all caused by clostridial extracellular protein toxins rather than by an immunological response to the bacterial infection itself or damage associated with bacterial growth in tissues (Madigan et al. Brock: Biology of Microorganisms. 12th ed. 2009, Pearson/Benjamin Cummings pp. 829-832).
3. Pseudomonas aeruginosa toxins. Pseudomonas aeruginosa is an opportunistic pathogen that secretes numerous extracellular proteins and enzymes that are virulence factors including: elastase, alkalin protease, a pore-forming cytotoxin, phospholipase C, and lechithinase (Pessi et al. (2001) J. Bac. 183:6676-6683). Once P. aeruginosa has colonized host tissue, these extracellular proteins and enzymes break down the host's physical barriers, damage host cells, and interfere with the host's immune response, collectively allowing disease to progress. P. aeurginosa also produces extracellular protein toxins (exotoxins) that mediate local and systemic disease processes.
4. Shiga toxins. Shigella dysenteriae, and the shigotoxigenis group of Escherichia coli (STEC), including serotype 0157:H7 and other enterohemorrhagic E. coli, produce large extracellular protein toxins that cause hemorrhagic colitis, kidney failure and death (Kaper and O'Brien, eds. Escherichia coli O 157:H7 and other Shiga Toxin-Producing E. coli Strains (1998), ASM Press (Herndon, Va.); and Frasher et al. (2006) J. Biol. Chem. 279:27511-27517). Shiga toxins inhibit protein synthesis by interfering with host ribosomes.
These are but a few examples of extracellular proteins and enzymes secreted by bacteria into tissues. Another extracellular enzyme is an iron reductase that is responsible for reducing Fe(III)→Fe(II), which is then bound and transported into the cell (Cowart (2002) Archives of Biochemistry and Biophysics, 400:273-281). Because iron is a required for bacterial growth, an extracellular enzyme that supplies iron to the infectious bacterium is important for bacterial growth.
Despite these examples, the majority of extracellular proteins and enzymes have functions that have not yet been characterized. Furthermore, little is known about the collective function of bacterial extracellular components or, more specifically, their function in terms of pathogenesis.
Vaccines
One mechanism used to halt the onset of pathogenesis and prevent disease is to improve host immunity by administering a biological preparation called a “vaccine” that establishes or improves immunity to a particular pathogen through the induction, or elicitation of an immunological response. Vaccines can be prophylactic (e.g., to prevent or ameliorate the effects of a future infection by any natural or “wild” pathogen) or therapeutic (e.g., vaccines against cancer, which are under investigation). Vaccines may comprise dead or inactivated microorganisms or purified products derived there from.
There are several types of vaccines currently in use, and follow different strategies to reduce risk of illness, while retaining the ability to induce a beneficial immune response. For instance, some vaccines contain killed microorganisms. These are previously virulent microorganisms which have been killed using chemicals or heat. Examples include vaccines against flu, cholera, bubonic plague and hepatitis A. Other vaccines contain live, attenuated microorganisms that have been cultivated under conditions that disable their virulence determinants. Alternatively, vaccines can comprise microorganisms that are closely-related, but less pathogenic than the organisms that cause the disease to produce a broad immune response. They typically induce infections and immunological responses without causing appreciable disease and are the preferred type of vaccines for use in healthy adults. Examples include vaccines for yellow fever, measles, rubella and mumps. The live attenuated tuberculosis vaccine called BCG, originally derived from cows, is immunogenic but does not cause extensive immunopathology.
Toxoids are inactivated toxic compounds, which are used in cases where the toxic compound, rather than the microorganism itself, cause illness. For example, the diphtheria vaccine is comprised of either formalin-treated diphtheria toxin or a recombinant toxin; neither is infectious, both stimulate the host's immune system to produce antibodies (Lobeck et al. (1998) Infection and Immunity. 66:418-423).
Vaccines need not contain a whole microorganism. Rather, a protein fragment of such microorganism can be used to illicit an immune response. Examples of such protein fragments used for vaccination are the surface proteins, suitably protein subunits, of Hepatitis B virus produced in yeast. Surface proteins with various functions are associated with the outer layer of both Gram-negative and Gram-positive bacteria. Many bacteria have mechanisms that impair antibody production by inducing suppressor cells, blocking antigen processing, and inhibiting lymphocyte mitogenesis. Some bacteria, such as, Neisseria gonorrhoeae, Haemophilus influenzae, Proteus mirabilis, clostridial species, and Streptococcus pneumoniae produce IgA-specific proteases that cleave and inactivate secretory IgA on mucosal surfaces. Other bacteria, such as pneumococci, meningococci, have capsules that prevent opsonic antibodies from binding. Typically protein or protein subunit vaccines are prepared using either surface proteins, external polysaccharides, or intracellular proteins.
For example, Staphylococcus aureus vaccines are presently available in the form of inactivated highly encapsulated S. aureus cells. Their efficiency for long-term treatment of disease, such as mastitis, has not been confirmed, and there is considerable variability in the structure of capsular polysaccharides which could limit the usefulness of this approach.
With respect to other bacteria of pathogenic importance for mammals vaccines comprising immunogenic virulence proteins are important. Such protein-based vaccines should be of particular value in the case of vulnerable subjects such as very young children, which are able to produce antibodies against foreign proteins. While bacterial proteins have been used to create vaccines, those proteins were intracellular or membrane bound, not secreted proteins. Accordingly, it is desirable to prepare bacterial vaccines constructed with at least one or more extracellular proteins to confer immunity against microorganism-associated infectious disease.